Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.     

Effect of 13-hydroperoxyoctadecadienoic acid on the supply of arachidonic acid for prostaglandin synthesis from arachidonoyl-CoA mediated by the cytosolic or microsomal acyl-CoA hydrolase in rabbit kidney medulla

The effects of 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the cytosolic or microsomal acyl-CoA hydrolase (ACH) activity in rabbit kidney medulla and on the ACH-mediated prostaglandin (PG) formation from arachidonoyl-CoA (AA-CoA) were examined. 13-HPODE (10, 20, and 50 microM) had no effect on the cytosolic ACH activity but significantly inhibited the activity of the microsomal enzyme (43-57% inhibition). PG formation was measured as follows: AA-CoA (20 nmol) was preincubated with the cytosolic or microsomal fraction (as the source of ACH) in the presence or absence of 13-HPODE for 5 min at 37 degrees C, followed by incubation with the microsomal fraction (as the source of PG-synthesizing enzymes), hydroquinone and reduced glutathione for 5 min at 37 degrees C, and the PGs formed were measured by HPLC, with use of 9-anthryldiazomethane for derivatization. 13-HPODE reduced the PG formation when the microsomal fraction, but not the cytosolic fraction, was used as the source of ACH (10, 20, and 50 microM; 28-55% inhibition). These results suggest that 13-HPODE may modulate PG levels in rabbit kidney medulla by inhibiting the microsomal ACH activity.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

A mutant with constitutive sexual organ development in<Emphasis Type="Italic"> Marchantia polymorpha</Emphasis> L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.     

A general computational model of mitochondrial metabolism in a whole organelle scale

SUMMARY: A computational tool for mitochondrial systems biology has been developed as a simulation model of E-Cell2, a publicly available simulation system. The general model consists of 58 enzymatic reactions and 117 metabolites, representing the respiratory chain, the TCA cycle, the fatty acid beta-oxidation and the inner-membrane transport system. It is based on previously published enzyme kinetics studies in the literature; we have successfully integrated and packaged them into a single large model. The model can be easily extended and modified so that mitochondrial biologists/physiologists can integrate their own models and evaluate them in the context of the whole organelle metabolism. AVAILABILITY: The mitochondrial model is bundled up with E-Cell2 simulation system, which can be downloaded from http://www.e-cell.org. CD-ROMs are also available and are distributed at major conferences. SUPPLEMENTARY INFORMATION: All the kinetic data are available via http://www.e-cell.org     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.